Large Differences in Aging Phenotype between Strains
of the Short-Lived Annual Fish Nothobranchius furzeri
Eva Terzibasi1,2., Dario Riccardo Valenzano3,4., Mauro Benedetti5, Paola Roncaglia5, Antonino
Cattaneo5,6 , Luciano Domenici1,7, Alessandro Cellerino2,3*
1 Istituto di Neuroscienze del CNR, Pisa, Italy, 2 Biology of Ageing, Fritz-Lipmann Institute for Age Research, Leibniz Institute, Jena, Germany, 3 Scuola Normale Superiore,
Pisa, Italy, 4 Department of Genetics, Stanford University, Stanford, California, United States of America, 5 International School of Advanced Studies, Trieste, Italy,
6 European Brain Research Institute, Rome, Italy, 7 School of Medicine, University of L’Aquila, Aquila, Italy
Abstract
Background: A laboratory inbred strain of the annual fish Nothobranchius furzeri shows exceptionally short life expectancy
and accelerated expression of age markers. In this study, we analyze new wild-derived lines of this short-lived species.
Methodology/Principal Findings: We characterized captive survival and age-related traits in F1 and F2 offspring of wildcaught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neurodegeneration at age 21 weeks.
Median lifespan in the laboratory varied from to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data
demonstrate that rapid age-dependent decline and short lifespan are natural characteristics of this species. The N. furzeri
distribution range overlaps with gradients in altitude and aridity. Fish from more arid habitats are expected to experience a
shorter survival window in the wild. We tested whether captive lines stemming from semi-arid and sub-humid habitats
differ in longevity and expression of age-related traits. We detected a clear difference in age-dependent cognitive decline
and a slight difference in lifespan (16% for median, 15% for maximum lifespan) between these lines. Finally, we observed
shorter lifespan and accelerated expression of age-related markers in the inbred laboratory strain compared to these wildderived lines.
Conclusions/Significance: Owing to large differences in aging phenotypes in different lines, N. furzeri could represent a
model system for studying the genetic control of life-history traits in natural populations.
Citation: Terzibasi E, Valenzano DR, Benedetti M, Roncaglia P, Cattaneo A, et al. (2008) Large Differences in Aging Phenotype between Strains of the Short-Lived
Annual Fish Nothobranchius furzeri. PLoS ONE 3(12): e3866. doi:10.1371/journal.pone.0003866
Editor: Jean-Nicolas Volff, Ecole Normale Supérieure de Lyon, France
Received July 21, 2008; Accepted October 29, 2008; Published December 4, 2008
Copyright: ß 2008 Terzibasi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by LayLineGenomics S.p.A., Scuola Normale Superiore, and the Leibniz Gemeinschaft. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: acellerino@fli-leibniz.de
. These authors contributed equally to this work.
scarce and erratic precipitation [6]. The current laboratory strain,
named GRZ, directly stems from this collection and was
maintained as a pure line by dedicated hobbyists. A conservative
estimate of 6 months generation-time implies 80 captive
generations, suggesting high levels of inbreeding (confirmed by
measurement of the homozygosity of polymorphic loci by
Reichwald et al., submitted).
We studied GRZ and recorded a median lifespan of 9 weeks
and a maximum of 12 weeks in the laboratory. This short lifespan
is coupled to fast growth and accelerated expression of age-related
phenotypes [2,7]. The lifespan of N. furzeri GRZ can be prolonged
by decreasing the water temperature [8] and by addition of the
natural compound resveratrol to food [9].
Classical evolutionary theories of aging predict that senescence
evolves as a result of the decreasing force of natural selection at
later ages. These theories predict that populations experiencing
low mortality due to external causes evolve retarded onset of
senescence [10–13]. Later work has questioned the generality of
these theories [14–16] and experimental tests have provided mixed
results. Increasing mortality induces evolution of rapid maturation
Introduction
Research into aging in vertebrates is hampered by the lifespan
of available model systems and tractable laboratory species with a
lifespan of less than 1 year are highly desirable [1].
Annual fishes of the genus Nothobranchius are a clade of teleosts
found in ephemeral bodies of water that form during the monsoon
season in eastern and southern Africa. All surviving adults die
when the habitat dries out and their maximum natural lifespan is
limited to several months, making them among the shortest-lived
vertebrates [2]. As an adaptation to the seasonal disappearance of
their habitat, they produce desiccation-resistant eggs that can
survive for one or several years encased in the dry mud in a
dormant state in which all biological processes are depressed
(diapause) [3–5]. Owing to their short lifespan, different species of
annual fishes were proposed as model systems for aging research
by several groups (reviewed by Genade et al., [2]).
Our studies focussed on the species Nothobranchius furzeri, which
was originally collected in 1968 in a seasonal pan of the Gona Re
Zhou (GRZ) National Park in Zimbabwe, a semi-arid area with
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other related, parapatric/allopatric species which are still
undescribed. The N.melanospilus/N.hengstleri species complex defines
a third clade and species form coastal Tanzania defines a forth
clade. Deeper relationships between these clades could not be
resolved and the sister clade to the N.furzeri/N.orthonotus species
complex remains to be determined.
Populations of N. furzeri showed a significant level of genetic
differentiation depending on their geographic origin. Three
samples collected in the lower Limpopo basin clustered and were
separated from three samples collected in a more northern area
closer to the border between Mozambique and Zimbabwe (Fig. 2).
This separation is supported by very high bootstrap values. The
average sequence divergence between northern and southern
populations in the analyzed region of the cox1 locus is 4.8%. These
data indicate that populations of the lower Limpopo basin are
isolated from more northern populations.
and faster senescence in Drosophila [17] and a comparison of
longevity in chemically protected (venomous) vertebrates as
compared to non-venomous sister taxa also supports the theory
that reduced predation induces evolution fo retarded senescence
[18]. However, a systematic study of natural populations of the
small tropical teleost guppy (Poecilia reticulata) originating from
high- and low-predation habitats did not detect any difference in
longevity or span of the reproductive period [19]. For annual fish,
the duration of ephemeral pans imposes an upper limit to the
natural life expectancy of different populations and species of
annual fishes, as adults cannot survive desiccation of their
environment. Fish inhabiting habitats with shorter duration of
seasonal water (i.e., more arid) may experience a shorter window
of survival than fish derived from more humid habitats.
To investigate the association of life-history traits and variables
related to the duration of seasonal water, we sampled the Limpopo
River drainage system in southern Mozambique in 2004. This
fluvial system presents a cline of altitude and annual precipitation/
evaporation on a small geographical scale and we succeeded in
establishing captive populations originating from different habitats
along this cline. Here, we report the genetic structure of N. furzeri
and the life-history characteristics of these wild-derived isolates.
Captive lifespan of F1 and F2 generations
Individuals collected from localities MZM-04/02, MZM-04/03
and MZM-04/06 were used as founders for new wild-derived
lines. Fishes collected from locality MZM-04/10 were separated
into three groups and became founders of three independent lines
named MZM-04/10P (founders 1 male and 1 female), MZM-04/
10T (founders 1 male and 2 females) and MZM-04/10G (founders
3 males and 5 females).
The survival of the F1 captive generation was recorded for MZM04/03, MZM-04/06 and MZM-04/10P. The number of individuals
followed over their lifespan is limited because F1 individual were
intended for breeding and not for analysis of life-history traits. All
isolates showed a lifespan at least double that of the laboratory strain
GRZ (Fig. 3A). The lifespan of the F1 captive generation of isolates
MZM-04/02, MZM-04/10T and MZM-04/10G could not be
recorded owing to technical problems in the fish facility.
Analysis of lifespan in the F2 generation focussed on MZM-04/
10 and MZM-04/03 (Fig. 3B) as representatives of northern and
southern populations. Inspection of eggs after 2 months of
incubation revealed the presence of developed embryos and these
were then hatched. The F2 generation of MZM-04/10P, MZM04/10G and MZM-04/10T isolates showed similar survivorship
characteristics and were pooled. The median lifespan of this
pooled group was 20 weeks, with 10% survivorship at 23 weeks.
The median lifespan of the MZM-04/03 isolate was 23 weeks,
with 10% survivorship at 29 weeks. These values were within the
ranges recorded for the F1 generation of the same populations.
Differences in longevity between F2 MZM-04/03 and pooled
MZM-04/10 are statistically significant (log rank test, P,0.05).
Unexpectedly, a strong effect of incubation time on post-hatch
lifespan was detected in the F2 generation of at least one isolate.
Inspection of F2 eggs of the MZM-04/10P isolate revealed the
presence of many embryos still vital after 12 months of incubation.
These were hatched in four different ‘‘cohorts’’ and they all
showed an extremely short lifespan similar to the GRZ laboratory
strain. The pooled data are reported in Fig. 3B. This isolate was
named MZM-04/10Plate. By contrast, attempts to hatch F2 eggs of
the MZM-04/03 isolate after 12-month incubation resulted in
only seven vital embryos. These developed into longer-lived
individuals than the late-hatched MZM-04/10Plate (age at death
10–15 weeks). At this stage, the study was interrupted because the
junior authors (A.C., E.T., D.V.) had to relocate for lack of
funding and the fish colony was moved from SNS Pisa to FLI in
Jena. Analysis of subsequent generations performed in Jena
revealed that the extremely short-lived phenotype observed in
the F2 generation of MZM-04/10Plate is not genetically fixed
(Supplementary figure S3).
Results
Collection of wild N. furzeri
In year 2004, we collected N.furzeri form several habitats in
Mozambique, South of the original collection point in the Gona
Re Zhou National Park in Zimbabwe (Fig. 1A). In particular, four
populations were used in this study: MZM-04/02, MZM-04/03,
MZM-04/06 and MZM-04/10 (for details on naming conventions
see Methods). Localities MZM-04/02 and MZM-04/03 are close
to the Limpopo River, localities MZM-04/06 and MZM-04/10
are in a system of intermittent (ephemeral) streams (Chingovo
system) originating on the plateau of the Gona Re Zhou National
Park in Zimbabwe. These streams do not reach the sea and
disappear in a series of inland shallow lakes and swamps [6]. These
two pairs of populations are therefore geographically isolated from
each other.
The area sampled presents a gradient of altitude and precipitation. Altitude decreases from 400 m in GRZ to 120 m at MZM-04/
10 to almost sea level at MZM-04/03 (Fig. 1B). Fig. 1C shows a GIS
interpolation map of the ratio between evaporation and annual
rainfall in the area of interest. An aridity cline is clearly visible.
Northern populations (GRZ, MZM-04/06, MZM-04/10) originate
from a typical semi-arid habitat, whereas southern populations
(MZM-04/02, MZM-04/03) originate from habitats that can be
considered as a transition to sub-humid. Table 1 summarizes the
principal habitat characteristics for these isolates.
Genetic structure of Nothobranchius wild populations
A partial sequence of the mitochondrial locus cox1 was used to
define the relationship between N. furzeri and its sympatric/
parapatric species, as well as to test for significant genetic
differentiation between geographically-separated populations of
N. furzeri. The data set included, in addition to samples collected by
us, one N. furzeri specimen originating from a 1999 collection
(Wood, 2000) and one from a 2004 collection (Brian Watters,
unpublished), as well as several specimens of all known species
from Mozambique and some species from coastal Tanzania.
The analysis revealed that N. furzeri forms a well-supported clade
that also contains its sympatric species N. orthonotus and the
parapatric species N. kunthae. This clade is separated from a second
clade with similar distribution range that contains N. rachovii and
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Figure 1. Location of N. furzeri habitats. (A) GPS of habitats mapped using GPS Visualizer onto a GoogleEarth image. Red flags represent habitats
for lines for which lifespan data were recorded. Save and Limpopo indicate the two major rivers that limit the distribution area. Small arrows point to
intermittent streams originating on the Gona Re Zhou plateau. Black flags represent habitats for which no lifespan data were recorded, but
specimens were included in the phylogenetic analysis. (B) The same habitats localized onto a GIS elevation map. (C) The same habitats localized onto
a GIS interpolation of the ratio between evaporation and 30-year annual average precipitation. Meteorological stations used for GIS interpolations are
indicated in black.
doi:10.1371/journal.pone.0003866.g001
precluding further analysis. A new collection trip is programmed
to obtain new breeding stock from this locality
Maximum likelihood estimates (MLE) of the demographic
parameters for three F2 isolates and the inbred laboratory stain
GRZ were obtained using WinModest software. These are
A small number (n = 11) of F2 MZM-04/06 individuals were
also followed. The data are reported in Fig. 3B. A discrepancy in
the lifespan of F1 and F2 generations was observed for this isolate
as well. This isolate proved difficult to breed and was lost as a
consequence of the relocation from CNR in Pisa to FLI in Jena,
Table 1. Summary describing the isolates used in the study.
Population
Year of collection
Elevation
Drainage system
GRZ
1969
,400 m
Chingovo
Habitat type
Semi-arid
MZM-04/10
2004
,120 m
Chingovo
Semi-arid
MZM-04/06
2004
,120 m
Chingovo
Semi-arid
MZM-04/03
2004
,50 m
Limpopo
Transition to sub-humid
MZM-04/02
2004
,50 m
Limpopo
Transition to sub-humid
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Figure 2. Distance-based cladogram of Nothobranchius from southern Africa. The cladogram is based on a partial sequence of the cox1
mitochondrial locus. The codes after the species names refer to specific collection points. The brackets indicate the geographic origin of the clade.
ZIM, Zimbabwe; MOZ, Mozambique, TAN; Tanzania. The support values over each node are the confidence probability obtained by the Minimum
Evolution algorithm. The support values under each node corresponds to the Neighbor Joining algorithm. The left values corresponds to interiorbranch test [40] and the right values to 3000 bootstratps. All trees used maximum composite likelihood (MCL) Computations were performed using
MEGA 4.0 [38].
doi:10.1371/journal.pone.0003866.g002
Figure 3. Survivorship of F1 and F2 generations. (A) Survivorship of the F1 generation of wild-derived fish MZM-04/03 (n = 8), MZM-04/06
(n = 28) and MZM-04/10P (n = 18). (B) Survivorship of the F2 generation of MZM-04/03 (n = 24), MZM-04/06 (n = 11), MZM-04/10G (n = 47) and MZM-04/
10P (n = 90) and of the inbred line GRZ (n = 93).
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Age-related markers: behavior
presented in Table 2. Differences in lifespan are accounted for by
differences in the demographic rate of aging (the b parameter of
the model). This parameter is almost six-fold higher for the GRZ
strain compared to the MZM-04/03 isolate. MLE of baseline
mortality (the a parameter of the model) showed large confidence
intervals, and differences among isolates were not statistically
significant.
Aging in the inbred GRZ strain of N. furzeri is associated with
reduced open-field exploration [23]. Open-field exploration was
quantified at 5 weeks and 9 weeks of age in GRZ and in F2
progeny of MZM-04/10G and MZM-04/3 strains. Less open-field
exploration was observed only in the short-lived GRZ strain. On
the other hand, the MZM-04/03 line showed an increase in
exploratory activity in this time window. (Fig. 9)
Age-dependent impairment of learning and memory is a
hallmark of aging in complex organisms and is observed in many
model systems, including Drosophila [24]. Age-dependent cognitive
decline was also recently described in zebrafish [25]. A form of
age-dependent learning decline is observed in the GRZ strain
between 5 and 9 weeks of age [23] and can be detected using a
protocol of active avoidance in a modified version of the shuttle
box test. We quantified age-dependent learning impairment in F2
progeny of MZM-04/10Plate, MZM-04/10G, MZM-04/3 and
MZM-04/02 isolates. MZM-04/10Plate and MZM-04/10G
showed a marked decrease in learning performance between 5
and 9 weeks of age that is reminiscent of the decline described in
the GRZ strain. The MZM-04/02 and MZM-04/03 lines showed
lower performance at 5 weeks of age compared to MZM-04/
10Plate and MZM-04/10G. However, their learning performance
did not decrease further at 9 weeks of age (Fig. 10). Therefore, the
age-dependent decline in conditioning did not correlate with
captive longevity, but with the geographic origin of the animals
tested.
Age-related markers: histology
We investigated the expression of age-related histological
damage in wild-derived lines and compared them with the
short-lived inbred strain GRZ. Lipofuscin is an auto-fluorescent
pigment that accumulates over time in a large variety of organisms
[20]. We previously reported rapid accumulation of lipofuscin in
the liver of the GRZ inbred strain [2,8]. Fluoro-Jade B is a general
histochemical marker of neurodegeneration [21] which was
observed during aging of the GRZ inbred strain [9]. Reduced
lifespan in the GRZ strain and in the long-incubation line MZM04/10Plate was coupled to accelerated expression of age-related
markers in both liver and brain compared to the F2 generation of
MZM-04/10G and MZM-04/3 (Fig. 4A,B).
We repeated the analysis of brain Fluoro-Jade B and liver
lipofuscin in FLI in Jena after relocation on GRZ and F6
generation of MZM-04/03 using specimens bred in Jena. In
addition, we analyzed expression of brain lipofuscin. Three brain
areas were analyzed separately: telencephalon, optic tectum and
hindbrain. Expression of liver lipofuscin was higher in the GRZ
strain at 11 weeks compared to age-matched MZM-04/03 fishes,
but was significantly lower than in 21-week-old MZM-04/03
(Fig. 5). Lipofuscin accumulation was also accelerated in the brain
of GRZ strain (Fig. 6), a pattern paralleled by faster expression of
Fluoro-Jade B positivity (Fig. 7). This acceleration affected the
three brain areas equally. Quantification of both age markers in
the brain revealed that 11-week-old GRZ individuals exhibited a
level of expression comparable to that in 21-week-old MZM-04/
03 individuals (Fig. 8).
It should be noted that several old specimens of wild-derived
lines showed a macroscopic ‘‘aged’’ phenotype that comprises
spinal curvature, emaciation and (in males) loss of color
(Supplementary material, Fig. S2). This age-related phenotype is
described in other fish species such as the guppy (Poecilia reticulata),
the South American annual fish Austrolebias bellottii and zebrafish
[22]. Although a considerable degree of variation is observed, fish
from wild-derived lines may spend several weeks in this ‘‘decrepit’’
state before eventually dying. This is not the case for fish of the
GRZ strain, which normally die before developing a macroscopic
phenotype.
Discussion
We characterized longevity and age-associated histological and
behavioral phenotypes in the F2 offspring of wild-derived N. furzeri
collected by the authors in 2004. In addition, we compared the
aging phenotype of captive lines derived from two habitats
differing in aridity and found that age-related cognitive deficit was
influenced by the geographic origin of the captive lines.
We measured the longevity of F1 and F2 offspring of fishes
collected from three different habitats differing in altitude and
evaporation/precipitation ratio. These out-bred populations
showed median longevity of 20–23 weeks and maximum longevity
of 25–32 weeks. These data, in combination with analysis of
histological age markers, demonstrate that wild-derived outbred
individuals of N. furzeri exhibit fast age-dependent decay and a
short lifespan. Rapid aging is therefore a natural trait for this
species.
Previous studies of the highly homozygous GRZ strain reported
a median lifespan of 9 weeks and a maximum lifespan of 13 weeks.
The large difference in lifespan between GRZ and wild-derived
Table 2. Maximum likelihood estimates of demographic parameters.
Population
Model
a
b
26
s
GRZ
Logistic
0.00012 (3.75610
1.01915 (0.60581–1.71452)
1.12696 (0.31687–4.00813)
MZM-04/10Plate
Logistic
0.00034 (0.00003–0.00385)
0.91556 (0.59512–1.40853)
1.22287 (0.46044–3.24778)
MZM-04/10G
Gompertz
0.00159 (0.00049–0.00509)
0.24433 (0.18981–0.31451)
–
MZM-04/03
Gompertz
0.00275 (0.00067–0.01126)
0.17105 (0.11756–0.24889)
–
20.00365)
The best-fitting model was selected by comparing the likelihood of four possible models: Gompertz, Gompertz-Makeham, logistic, and logistic-Makeham. The logistic
model provided the best fit of the GRZ and MZM-04/10Plate data (likelihood ratio test, P,0.01), but not for MZM-04/10G and MZM-04/03, for which the simpler Gompertz
model was used. The a parameter represents baseline mortality, the b parameter represents demographic rate of aging, and the s parameter is the deceleration of the
logistic model. Values in parentheses represent 95% confidence intervals. All simulations were performed using WinModest.
doi:10.1371/journal.pone.0003866.t002
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Figure 4. Age-dependent histological markers. (A) Representative confocal images depicting lipofuscin staining in the liver (upper row) and
Fluoro-Jade B staining in the optic tectum (lower row) in the different strains at two ages. (B,C) Quantification of lipofuscin autofluorescence and
Fluoro-Jade B staining as a percentage of the threshold area. Sample size n = 3 for all strains and ages. Error bars represent standard error of the
mean. Student’s t-test, *P,0.05, **P,0.01.
doi:10.1371/journal.pone.0003866.g004
lines is certainly accounted for in part by inbreeding depression, as
analysis of polymorphic markers has demonstrated that the GRZ
strain is extremely homozygote (Reichwald et al., submitted).
However, other factors related to the captive history of the strain
and its original habitat may contribute to this accelerated aging
phenotype.
In mice [26] and Drosophila [27–29], involuntary selection in
captivity for high productivity under benign conditions leads to
rapid evolution of a shorter lifespan, an effect that is distinct from
inbreeding depression. Such captive selection might have
influenced the GRZ strain as well.
This study was designed to test the hypothesis that ecological
variables associated with the duration of water and population
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isolation in the natural habitat of N. furzeri lead to evolution of
different aging rates and longevity between fish strains derived
from different regions.
The combined differences in altitude and evaporation/precipitation ratio influence the duration of the temporary habitat, since
intermittent stream are drained and water accumulates in
downstream habitats after the rains. Moreover, the precipitation/evaporation ratio directly influences a meteorological variable termed ‘‘length of the growing period’’, which is the number
of days in the year when precipitation is higher than evaporotranspiration. The length of the growing period is usually discussed
in the context of water availability for crops, but it clearly also
influences the duration of seasonal ponds.
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Figure 5. Lipofuscin in the liver. Confocal images taken at an excitation wavelength of 488 nm. Images are projections of seven confocal planes
at a distance of 1 mm. (A) Liver section from 21-week-old MZM-04/03. (B) Liver section from 11-months old MZM-04/03; white arrowheads point to
autofluorescent erythrocytes, which were excluded from the analysis. (C) Liver section from 11-week-old GRZ; white arrowheads point to
erythrocytes, which were excluded from the analysis. (D) Quantification of lipofuscin density based on percentage threshold area. Student’s t-test,
*P,0.05, **P,0.01.
doi:10.1371/journal.pone.0003866.g005
We could indeed demonstrate large genetic differences across
different lines exist, which set the basis for a QTL analysis of
longevity in this species (see below). Support for the life-history
hypothesis, is inconclusive at this stage, however. We plan to
collect and characterize more wild-derived N. furzeri isolates,
including animals collected in GRZ as well as in the very humid
habitats North of the Save River. We also plan to study the two
sympatric species N. orthonotus and N. sp. aff. rachovii, to test for
parallel evolution of life-history traits in different species of the
genus Nothobranchius.
It is however to report that a note on breeding N. furzeri GRZ
dating back to 1973 [30], 4 years after collection of the founders,
already described a relatively short-lived phenotype (the breeding
pair showed typical hunchback at 3.5 months of age and lived for
4.5 months), suggesting that alleles conferring a short-lived
phenotype were present in the founder population and were fixed
early during the captive history of the GRZ strain.
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Although the correlation between captive lifespan and ecological conditions of the original habitat is intriguing, evolution of the
short-lived phenotype in the GRZ strain can only be elucidated by
studying the offspring of fish collected from the terra typica in GRZ
as well as N.orthonotus, which is found in the same habitat.
We analyzed two different age-related markers: lipofuscin in
brain and liver and Fluoro-Jade B in the brain. Lipofuscin is an
autofluorescent pigment that accumulates with age in many
animal models and is considered a robust age marker [20,31]. We
observed that lipofuscin accumulation was faster in the short-lived
GRZ strain than in the longer-lived MZM-04/03 wild-derived
line. This observation demonstrates that the short lifespan of the
GRZ strain is coupled to faster histological aging. However,
individuals from wild-derived lines can spend several weeks in a
highly degenerate ‘‘decrepit’’ state characterized by exaggerated
spinal curvature, weight loss and extreme emaciation before death.
Age markers demonstrated that the aging process is accelerated in
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Figure 6. Lipofuscin in the brain. Confocal images taken at an excitation wavelength of 488 nm. Images are projections of seven confocal planes
at a distance of 1 mm. (A) Telencephalon, (B) optic tectum, and (C) hindbrain from 21-week-old MZM-04/03. (D) Telencephalon, (E) optic tectum, and
(F) hindbrain from 11-week-old MZM-04/03. (G) Telencephalon, (H) optic tectum, and (I) hindbrain from 11-week-old GRZ. White arrows denote
lipofuscin granules. Arrowheads point to autofluorescent erythrocytes, which were excluded from the analysis.
doi:10.1371/journal.pone.0003866.g006
the GRZ strain, but comparison of captive longevity might
overestimate the magnitude of this difference. Direct comparison
of the timing of lipofuscin accumulation also pointed to differences
in aging rates across organs. Brains of 11-week-old GRZ
individuals showed levels of lipofuscin accumulation and FluoroJade-B staining comparable to those in 21-week-old MZM-04/03
individuals. However, livers from 21-week-old MZM-04/03
individuals exhibited significantly higher levels of lipofuscin
accumulation than those from 11-weeks-old GRZ individuals.
These data suggest that age-dependent degeneration in the GRZ
strain is faster in the brain than in the liver.
We measured age-dependent cognitive decay in the GRZ
inbred strain and in four different wild-derived lines. The lines
MZM-04/10Plate and MZM-04/10G, derived from northern
habitats, showed an age-dependent pattern of decline in learning
performance similar to that previously reported for the GRZ
inbred strain: high performance at 5 weeks of age that decreased
by 9 weeks of age. It should be noted that F2 MZM-04/10Plate
individuals showed a short lifespan and accelerated expression of a
neurodegeneration marker, whereas the MZM-04/10G line
showed a maximum lifespan of 26 weeks. Learning decline was
not correlated with lifespan or generalized neurodegeneration. On
the other hand, the lines MZM-04/02 and MZM-04/03,
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geographically and genetically distinct from the MZM-04/10
populations, showed low performance at 5 weeks, but no further
decline by 9 weeks of age. Therefore, we could detect a clear
correlation between age-dependent learning decline and the
geographic origin of the lines. The difference in this age-related
trait could be a neutral event stemming from genetic separation
between northern and southern populations, or an evolutionary
response to different ecological conditions.
Classical evolutionary theories of aging predict that populations
experiencing lower mortality due to external causes evolve
retarded onset of senescence compared to populations with higher
extrinsic mortality [11–14]. This theory has so been subjected to
few experimental tests in vertebrates [18,32,33]. Recently, a
systematic study of natural isolates of the small tropical fish guppy
(Poecilia reticulata) experiencing high and low levels of predation
detected no effect of extrinsic mortality on the evolution of
longevity or reproductive lifespan, but did detect an effect on
functional parameters of aging [19]. The peculiar ecology of
annual fishes offers a complementary model system to study the
effects of extrinsic mortality on the evolution of aging. Owing to
large differences in the length of the growing period, pools in semiarid highland habitats dry out faster than pools in humid lowland
habitats. Assuming that pool duration is a proxy for the maximum
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Aging in Annual Fish
Figure 7. Fluoro-Jade B staining in the brain. Confocal images taken at an excitation wavelength of 488 nm. Images are projections of seven
confocal planes at a distance of 1 mm. (A) Telencephalon, (B) optic tectum, and (C) hindbrain from 21-week-old MZM-04/03. (D) Telencephalon, (E)
optic tectum, and (F) hindbrain from 11-week-old MZM-04/03. (G) Telencephalon, (H) optic tectum, and (I) hindbrain from 11-week-old GRZ. White
arrows denote labeled neuronal processes and arrowheads point to autofluorescent erythrocytes, which were excluded from the analysis.
doi:10.1371/journal.pone.0003866.g007
window of adult survivorship in the wild, fishes in more arid
habitats should experience a shorter maximum lifespan than fishes
in wetter habitats. Indeed, when a small sample of different species
of annual fish were compared over a broad geographic scale, an
indication for a correlation between meteorological conditions of
the original habitat and captive lifespan was observed [2]. Our
experiment was designed to test whether habitat differences can
influence the evolution of life-history traits within one species. N.
furzeri was selected because the distribution range of this species
overlaps with a cline in altitude and precipitation/evaporation. We
are unable to provide a definitive conclusion, as we could not
obtain wild fish from GRZ (Zimbabwe), i.e., the northern end of
this cline. We plan to further characterize aging phenotypes and
survival in more wild-derived N. furzeri isolates and in the two
sympatric species N. orthonotus and N. sp. aff. rachovii to test for the
effects of extrinsic mortality on the evolution of life-history traits in
Nothobranchius.
A clear difference was observed in age-dependent cognitive
decline between the southern MZM-4/02 and MZM-4/03 lines
and the northern populations, including the GRZ strain. Northern
populations exhibited higher performance at younger ages, but
faster cognitive decline at later ages compared to populations from
more humid habitats. These results are in line with previous
studies in natural populations of guppy (Poecilia reticulata), for which
populations subject to lower extrinsic mortality exhibited lower
escape performance at young ages, but a less steep age-dependent
decay [19]. Both our study and that by Reznick et al. (2004) suggest
PLoS ONE | www.plosone.org
that extrinsic mortality influences the evolution of functional lifehistory traits.
Different lines of N. furzeri exhibited large differences in lifehistory traits. Genome-wide scanning for quantitative traits in N.
furzeri could represent an alternative method for identification of
loci accounting for differences in longevity and age-related
phenotypes in natural populations. Genomic sequences of N.
furzeri are now available (Reichwald et al., submitted) and are
being used to derive polymorphic markers for linkage studies.
Analysis of backcrosses and F2 GRZ6MZM-04/03 hybrids is
expected to provide a picture of the chromosomic loci responsible
for the large differences in life-history traits we described here.
Methods
Fish collection
A form similar to N. furzeri was discovered in 1999 on an
amateur collection trip in the lower Limpopo River drainage
system in southern Mozambique. This habitat is located
approximately 300 km south of the original collection point in
the GRZ National Park [34]. The form is very similar in shape to
N. furzeri but has a red, rather than yellow tail (Supplementary
material, Fig. S1).
In the last week of March 2004, we sampled ephemeral pans in
southern Mozambique between the Save and Limpopo Rivers.
Fish were collected using fine-meshed hand-nets. Fish were bagged
one per bag and all water in the bag was changed every day.
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Aging in Annual Fish
Figure 8. Quantification of (A) Fluoro-Jade B and (B) lipofuscin staining in the brain as a percentage of the threshold area. Data for
the telencephalon, optic tectum and hindbrain are presented separately. Error bars represent the standard error of the mean. Significance is reported
only for comparison between GRZ 11 weeks and MZM-04/03 11 weeks. Student’s t-test, *P,0.05, **P,0.01.
doi:10.1371/journal.pone.0003866.g008
Individuals of N. furzeri were collected from eight habitats and
individuals from four habitats were successfully transported and
bred in captivity. All these habitats were pools and pans in the
vicinity of rivers with a muddy substrate. We established that the
form depicted by Wood [34] is a different color morph of N. furzeri.
The two morphs are distributed along a north–south gradient
(Supplementary material, Fig. S1) and co-exist with two other
species of Nothobranchius: N. orthonouts and N. sp. aff. rachovii [34]. A
Figure 9. Age-dependent decrease in exploratory activity. Exploratory activity was measured in an open-field setting [9] in terms of (A) mean
velocity and (B) time spent moving. Error bars represent standard error of the mean. Sample size n = 10 for each age and population. Pairwise
comparisons were performed exclusively within populations. Mann-Whitney U-test, **P,0.01, ***P,0.001.
doi:10.1371/journal.pone.0003866.g009
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Aging in Annual Fish
Figure 10. Age-dependent cognitive deficit. The average top scores of an active avoidance paradigm [9] are reported. Error bars represent
standard deviation. Rank-based ANOVA, **P,0.01; ns, not significant. Comparisons among strains at age 5 weeks are indicated by brackets. The
symbols on the 9-weeks bars refer to comparison between the two ages within each strain. Sample size is 10 animals for each age and strain.
doi:10.1371/journal.pone.0003866.g010
recent and more systematic surveys has largerly confirmed this
biogeographic pattern [35]. The locations of the collection points
are shown in Fig. 1A. Sampling was restricted to transects
corresponding to primary/secondary roads. Many more N. furzeri
habitats certainly exist in the area, but are not easily accessible.
According to the convention of Nothobranchius collectors, collection
points were named using a code containing the name of the
country, the year of collection and a progressive number
indicating the order of collection points along the transect. The
wild-derived populations analyzed in the present study were
named after their collection points: MZM-04/02, MZM-04/03,
MZM-04/06 and MZM-04/10. Localities MZM-04/02 and
MZM-04/03 Habitat characteristics are reported in Table 1
30 min. Chrimonous larvae in Pisa were purchased from EscheMatteo (Parma, Italy). Chrimonous larvae in Pisa were purchased from
Poseidon Aquakultur (Ruppichterot, Germany). Twice a week the
bottom of the tanks was siphoned and 50% of the water was
exchanged with tempered tap water.
Survival assay
Surviving fish were counted every week starting from the forth
week. Mortality in fishes younger than 4 weeks usually occurred in
the first days after hatching. Dead fish were not counted because
they decay fast in water and may be eaten by their tankmates before
they are noticed. To compute differences among treatments, we
used commercially available GraphPad and Origin programs.
Demographic analysis was performed using WinModest software (http://www.hcoa.org/scott/softw-winmodest.asp).
Meteorological analysis
GIS maps were computed using FieldPro v. 0.91. Precipitation,
altitude and evaporation data were obtained from the website
http://geonetwork3.fao.org/climpag/agroclimdb_en.php
and
monthly mean values are reported on graphs. Fig. 1 shows the
ratio of monthly normalized evaporation to monthly normalized
precipitation.
Histology and histochemistry
Fishes were euthanized with MS-222 and cooled on crushed ice
for 5 min before dissection. Target tissues were dissected and fixed
by immersion in 4% paraformaldehyde/0.1 M phosphate buffer
(pH 7.4). Fishes analyzed in Pisa were infiltrated with 30% sucrose
to ensure cryoprotection, embedded in Tissuetek (Reichart-Jung,
Nubloch, Germany), frozen at 220uC, and serially sectioned
(thickness 18 mm) using a cryostat. Fishes analyzed in Jena were
embedded in Paraplast and sections of 5 mm in thickness were cut.
Intracellular accumulation of lipofuscin during aging was
detected in brain and liver tissues from young and old fishes as
light blue autofluorescent granules under UV excitation. For
quantification, images were acquired using a Leica confocal
microscope (in Pisa) or a Zeiss LSM (in Jena) at an excitation
wavelength of 488 nm, with fixed confocal parameters (pinhole,
photo-multiplier, laser intensity, etc.).
Fluoro-Jade B staining [21] was used to visualize neurodegeneration in brain tissues. Neurofibrillary tangles in brain tissues from
young and old fishes were quantified by analysis of confocal images
acquired using the same procedure as for lipofuscin quantification.
Fish culture
Eggs were maintained on wet peat moss at room temperature in
sealed Petri dishes. When embryos had developed, eggs were
hatched by flushing the peat with tap water at 16–18uC. Embryos
were scooped up and transferred to a clean vessel. Fry were fed
with newly hatched Artemia nauplii for the first 2 weeks and then
weaned with finely chopped Chironomus larvae. Fishes were starting
in the fourth week of life, when they are considered sexually
mature, fish were moved to 40-l tanks at a maximum density of 20
fishes per tank. Filtration was provided with air-driven sponge
filters. The temperature was maintained at 25uC using stab heaters
(in Pisa) or by climatising the entire room (Jena). Light/dark cycles
were amntained 12:12 using a timer. Fish were fed twice a day
with frozen Chironomus larvae at a quantity that they consumed in
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Aging in Annual Fish
Fluorescence analysis of images for both lipofuscin and FluoroJade B staining was performed using Metamorph software.
and Cox1rev, CAA TAA TGG CAA ATA CTG C). DNA was
extracted from fin clips preserved in ethanol. PCR conditions were
as follows: 5 min at 94uC; 5 cycles of 94uC for 45 s, 43uC for 30 s,
and 72uC for 30 s; 25 cycles of 94uC for 45 s, 48uC for 30 s, and
72uC for 30 s; and a final 15 min at 72uC.
Amplicons were cloned in a pGEMTeasy vector and sequenced
in both directions. Sequences were aligned using the program
CLUSTAL W [37] followed by manual inspection and modification. Sequences have been deposited in GenBank under accession
numbers EF464684–EF464713. For analysis, the primer sequences were trimmed to leave a sequence of 437 bp. Sequences were
analyzed using MEGA 4.0 [38] with indels or ambiguities deleted
from the analyses. T he maximum composite likelihood [39] with
rate heterogeneity (C = 0.5) was used. We also used the Minimum
Evolution algorithm [40], Log(Det) distances [41] and the Kimura
2 correction parameter [42]. All methods retrieved the same
clades. Confidence probabilities [41] for branches of the NJ tree
were assessed using the interior-branch test implemented in
MEGA. We opted to use this method based on data suggesting
that the bootstrap conservatively underestimates the statistical
support for groupings within a topology, particularly when large
numbers of taxa are being analyzed [43]. Trees were rooted with
Pronothobranchius kiyawense, the species of a monotypic sister genus.
Open-field-like assay
Single fish were scored for locomotor activity in a 20-l test-tank at
the same temperature as the home tank. Video recordings were
made using a digital video camera from above and the water level
was kept very low to minimize displacement on the z-axis, which
would not be picked up by the camera while recording from above.
Fishes were allowed to habituate for 30 min within the tank before
the 10-min recording was started. Image analysis was performed
using Ethovision software (Noldus, Wageningen, The Netherlands)
to compute the mean maximum velocity and the percentage time
spent moving for each fish belonging to all experimental groups. For
every group, the mean and standard deviation were computed.
Active avoidance task
Active avoidance was measured using a modified version of the
shuttle box [9]. A tank (38 cm623 cm618 cm) was divided in two
by a hurdle with a rectangular hole (3 cm63 cm). The two
compartments were wedged-shaped to funnel a fish through the
hurdle. The tank was filled with water from the housing tank and
the fish was left to acclimate for 15 min before starting the test.
Then the conditioned stimulus (red light) was delivered in the
compartment where the fish was present, followed by an adverse
stimulus. The fish always responded to the disturbance by moving to
the other compartment. The aim of the test was to detect the
acquisition of a strategy to escape from the adverse stimulus by
crossing the hurdle upon presentation of the conditioned stimulus.
The conditioned stimulus lasted for 30 s. If the fish did not move to
the other compartment after 15 s, the adverse stimulus was
delivered for 15 s. The fish moved to the other compartment,
resting for 30 s, and then the cycle was repeated. If the fish crossed
the hurdle within 15 s (i.e., before the onset of the conditioned
stimulus), the trial was scored as ‘‘success’’, otherwise it was scored
as ‘‘failure’’. A complete session consisted of 50 consecutive trials.
Two indexes were scored to assess learning in each experimental group. The first measure was the performance index, which
was used to visualize the evolution of the performance as a
function of the trial. Successful trials were scored as 1 and failures
as 0. For each trial, we computed an average score; for example,
for trial 1 in n fishes, the average score is:
AS1 ~
Supporting Information
Distribution of N.furzeri color morphs
Found at: doi:10.1371/journal.pone.0003866.s001 (0.10 MB
PDF)
Figure S1
Macroscopic phenotype of senescent N.furzeri
Found at: doi:10.1371/journal.pone.0003866.s002 (0.03 MB
PDF)
Figure S2
Figure S3 Analysis of subsequent generations. The F3 generation
of the MZM-04/10Plate isolate could not be analyzed because the
authors (AC, ET, DV) had to relocate their laboratories and
establish new fish facilities. Subsequent generations were hatched
after 2–3 months of incubation, but their lifespan was not recorded
as animals were sacrificed for other purposes. A survivorship
analysis of MZM-04/10Plate was performed in Jena and results
were compared with GRZ raised in Jena (Fig. 9). Longevity of the
GRZ strain in Jena was substantially longer compared to Pisa, with
a median lifespan of 11.5 weeks and 10% survivorship at 15 weeks.
This difference is not unexpected given differences in water
chemistry and food source between the two sites. Analysis revealed
that the extremely short-lived phenotype observed in the F2
generation of MZM-04/10Plate is not genetically fixed. Median
lifespan of MZM-04/10Plate in Jena was 29 weeks, with 10%
survivorship at 41 weeks. A lifespan characterization of MZM-04/
03 line in Jena is yet to be completed.
Found at: doi:10.1371/journal.pone.0003866.s003 (0.63 MB TIF)
1 Xn
PðiÞ,
i~1
n
where P(i) is 0 or 1. The performance index (PI) was then obtained by
averaging 10 consecutive AS values from trials 10 to 50 and plotting
each score as follows: PI(10–50) = AS1–10, AS2–11, …, AS41–50. The
top score index (TSI) was measured as the mean of the highest scores
reached by individuals from one group during 50 trials. Compared
to PI, TSI provides an absolute measure of ability to succeed in a
task, independent of the trial. TSI is computed as the mean for all
individuals in an experimental group as:
Acknowledgments
We wish to thank Stefano Valdesalici for help in designing and performing
the collection trip, Miles Parisi for help during field work, Graeme Ellis and
Jeremy Baker for logistics, Tyrone Genade for assistance in the fish room,
and Christoph Englert and Mathias Platzer for providing support and
laboratory space.
X
X11
X50
1
10
max
PðiÞ,
PðiÞ, . . . ,
PðiÞ :
i~1
i~2
i~41
10
Author Contributions
Conceived and designed the experiments: ET DRV AC LD AC.
Performed the experiments: ET DRV MB PR. Analyzed the data: ET
DRV AC. Wrote the paper: ET DRV AC. Performed the field work: AC
ET DRV.
Molecular phylogeny
Primers for amplification of cox1 in Nothobranchius were those
reported by [36] (Cox1for, AAC ACC TAT TCT GAT TCT TT;
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Aging in Annual Fish
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